The shortage of available donor allografts continues to provoke a health care crisis and provides the rationale for continued xenotransplantation research. We are attempting to address these issues by inducing transplantation tolerance across discordant xenogeneic barriers in non-human primates. Our strategy utilizes composite vascularized thymic-renal grafts from GalT-KO swine. During the most recent project period (late 2004-2008), we markedly decreased the complication rate in the induction period with a modified regimen, which eliminated steroids and whole body irradiation. Our most significant findings are (1) Extended average survival of life-supporting kidney xenograft recipients to greater than 50 days under the modified immunosuppression regimen as compared to 33 days under the original protocol; (2) Reproducible in vitro evidence for donor-specific tolerance; and (3) Demonstration of CD4/CD8 double positive baboon xenothymocytes in transplanted pig thymic grafts, indicating baboon thymopoiesis, which had otherwise not been achieved in a large animal xenotransplant model. To our knowledge, these results represent the longest average survival of life-supporting xenografts and also provide novel in vitro evidence for tolerance in large animals. In order to make this approach more clinically applicable, this proposal will address remaining crucial concerns of tolerance induction, immune suppression, and issues with renal xenograft function in baboons that may be caused by molecular incompatibilities across species. In Aim 1, we will first confirm immunologic tolerance induced by xenogeneic porcine vascularized thymic grafts and eliminate maintenance immunosuppression using our most successful regimen. We will also attempt to achieve tolerance with a T-cell depletion based regimen in order to remove anfi-CD154 mAb and myelosuppressive drugs. In Aim 2, we will assess in-vitro parameters of xenogeneic immunity before and after transplantation of vascularized thymic tissue. We will examine the fundamental cellular processes involved in the development and maintenance of tolerance. In Aim 3, we will evaluate the cause of xenograft kidney proteinuria and develop preventive strategies. We will explore: 1) the utility of anti-non-Gal antibody removal prior to graft implantation; 2) pharmacologic adjuvant vascular protective therapies shown to be clinically effective for treatment of vascular injury/proteinuria; and 3) the use of kidneys from GalT-KO pigs with additional transgenes, including hDAF, hCD39 and thrombomodulin. The success of a clinically applicable strategy would provide not only a limitless supply of donor organs but also opportunities for transplantation in patients unable to receive a transplant because of presensitization to allogeneic human donors.